Description:

Objective:

The objective of this research project is to examine the relationship of pesticide
mixtures and immunotoxic risk in vitro, as it pertains to the
role of free radicals. Humans and other animals are exposed to a myriad of
chemicals, either concurrently or sequentially, throughout their lifetimes.
Often, the metabolism of these chemicals results in the formation of unstable,
free radical intermediates or reactive oxygen species (ROS). Studies have indicated
that these ROS play a role in chemical metabolism and immune-cell response
and regulation. The hypothesis is that ROS and/or other free radical intermediates,
generated during the metabolism of various pesticides, mediate the immunotoxic
effects of environmental toxicants in mixture.

Approach:

Our approach is first to determine the extent of
immune response by C57BL/6 young adult, male mice splenocytes, in vitro, to
three different
pesticides and mixtures of those pesticides. To do this, splenocytes are isolated
and treated with piperonyl butoxide (PBO), lindane, or malathion to obtain
time- and dose-response curves. Two assays have been chosen to assess the concentration
of each pesticide that yields approximately 25 percent immunotoxicity. The
alamarBlue Cytotoxicity Assay uses the fluorogenic redox indicator dye, alamarBlue,
to monitor metabolically active cells. The In Situ Cell Death Assay labels
nicks in the DNA of cells undergoing cell death, and is analyzed via flow cytometry.
After identifying the pesticide concentration for 25 percent cytotoxicity,
splenocytes can be treated with mixtures of two pesticides (PBO and malathion,
lindane and malathion, and each pesticide alone at one concentration).

Secondly, we will examine the role of ROS in immunotoxicity, by assessing
the levels of oxidative enzymes in splenocytes treated with pesticides and
mixtures of pesticides. Often, when cells or organisms are exposed to chemicals,
ROS are generated, and enzyme induction can offer cellular protection against
these ROS. Thus, oxidative enzyme levels can be used as indicators of the oxidative
status of cells. The enzymes of interest are superoxide dismutase, catalase,
glutathione peroxidase, and glutathione reductase. Quantitation of each enzyme
in the cytosolic fraction of the cells, treated with various pesticides, will
be monitored using colorimetric techniques.

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.